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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Feb - 13 Mar 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no radioactive or other labelling used for determination of cell proliferation, in pre-screen test not tested up to a dose level of 100%

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
yes
Remarks:
no radioactive or other labelling used for determination of cell proliferation, in pre-screen test not tested up to a dose level of 100%
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2008
Deviations:
yes
Remarks:
no radioactive or other labelling used for determination of cell proliferation, in pre-screen test not tested up to a dose level of 100%
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
adopted in 2003
Deviations:
yes
Remarks:
no radioactive or other labelling used for determination of cell proliferation, in pre-screen test not tested up to a dose level of 100%
Principles of method if other than guideline:
- Principle of test: modified LLNA without any labelling of lymphocytes for determination of cell proliferation
- Short description of test conditions: During tier 1 LLNA, three concentrations of test substance were applied on three consecutive days to the dorsum of both ears. Mice were killed 24 h after the last application to determine ear and lymph node (LN) weights and LN cell counts.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Chemical structure
Reference substance name:
2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide
Cas Number:
1211443-61-6
Molecular formula:
C14 H17 Cl N4 O
IUPAC Name:
2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Remarks:
inbred, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 18 - 21 g
- Housing: in groups in Makrolon cages (MIII type; height 18 cm) with sterilized sawdust bedding material and paper cage enrichment and shelters
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no, ears were intact and free from any abnormality

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Pre-screen test: 25 and 50% (w/w)
Main test: 0.5, 5 and 50% (w/w)
No. of animals per dose:
Pre-screen test: 2
Main test: 6
Details on study design:
PRE-SCREEN TEST:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. Four young adult animals were selected and two test item concentrations of 25 and 50% were tested, each on two animals. On Day 4 animals were sacrificed and skin was observed for signs of irritation including oedema. No skin irritation was observed in any of the animals examined up to the highest dose tested. Based on the results, the highest test item concentration selected for the main study was a 50% concentration.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA, non-radioactive method
- Criteria used to consider a positive response: The following thresholds were used: ear weight index: 1.05, LN weight: 1.2, LN cell count: 1.3.
Values which exceed these thresholds were considered positive with no statistical significance, but a clear concentration-dependence or when a statistically significant increase in one of the parameters occurs and a clear concentration-dependence can be derived. Values which are below these thresholds were considered positive when a statistical significance occurs in one of the parameters together with a clear concentration-dependence.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, positive or solvent control on Day 1. The application was repeated on Day 2 and 3. Approximately 24 hours after the last treatment, all animals were sacrificed by intra-peritoneal injection with pentobarbital. Both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 8 mm => 0.5 cm²). For each animal both punches were immediately weighed pooled per animal using an analytical balance after which the punches were discarded. Both auricular draining lymph nodes (left and right) of mice were excised. The relative sizes of the nodes (as compared to normal) were estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. For each animal both lymph nodes were pooled and immediately weight using an analytical balance. A single cell suspension (LNC) was prepared by gentle separation through a 200 µm mesh stainless steel gauze. LNC were collected in approximately 0.7 mL of PBS in a 24 wells plate that was kept on ice. Cell counts were determined using a Coulter Counter Z1 Dual (Beckman Coulter, The Netherlands).

IN-LIFE EXAMINATIONS:
Animals were observed for mortality/viability twice daily and for signs of toxicity once daily on Days 1-3 between 3 and 4 h after dosing. Subject appraisal of food and water consumption was maintained during the study period. Body weight was determined on Day 1 (re-treatment) and 4 (prior to necropsy). The ears were observed for skin reactions and irritation on Days 1-4 (on Days 1-3 within one hour after treatment) according to Draize.

INDEX CALCULATIONS:
Index of ear weight (IWear) of the test group were calculated as follows:
IWear = mean weight (MW)%ear / MW1ear

MW%ear : the mean weight of pooled ears of the test group in [mg]/0.5 cm² per pair of ears
MW1ear : the mean weight of pooled ears of the vehicle control group in [mg]/0.5 cm² per pair of ears

Index of lymph node weight of the test group (IW LN) were calculated as follows:
IW LN = MW%LN / MW1LN

MW%LN : the mean weight of pooled lymph nodes of the test group in [mg] per pair of lymph nodes
MW1LN : the mean weight of pooled lymph nodes of the vehicle control group in [mg] per pair of lymph nodes

Index of total lymph node cell-count of the test group (IC LN) were calculated as follows:
IC LN = MC%LN / MC1LN

MC%LN : the mean cell-count of pooled lymph nodes of the test group in [x 10E6 / mL] per pair of lymph nodes
MC1LN : the mean cell-count of pooled lymph nodes of the vehicle control group in [x 10E6 / mL] per pair of lymph nodes

EVALUATION CRITERIA:
- Skin irritation:
Weak: 5% ≤ Threshold concentration (TC)
Moderate: 0.1% ≤ TC < 5 %
Strong: TC < 0.1 %

- LN hyperplasia:
Weak: 5% ≤ TC
Moderate: 1% ≤ TC < 5 %
Strong: TC < 1 %

Evaluation citeria were derived from results obtained with standard irritants and contact allergens (Ulrich P, Streich J, Suter W (2001).

Reference: Ulrich, P, Streich, J and Suter, W., Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals. Arch. Toxicol. 74: 733-744, 2001.

Positive control substance(s):
other: 1-chloro-2,4-dinitrobenzene (DNCB) (0.5%)
Statistics:
In case of a positive finding as described above, a linear regression was used to calculate the concentration corresponding to the respective threshold indices. The calculation for the LN hyperplasia threshold concentration was performed with the respective LN cell count threshold index. In case of inconclusive cell count results, LN weight data were used to calculate the threshold concentration. Calculations were performed in MS EXCEL and statistical analysis was performed with GraphPad Prism 4 (Kruskal-Wallis test, followed by the Mann Whitney test).

Results and discussion

Positive control results:
The positive control DNCB elicited a reaction pattern with increased LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
other: ear weight index
Value:
1
Test group / Remarks:
0.5%
Key result
Parameter:
other: local lymph node weight index
Value:
1.14
Test group / Remarks:
0.5%
Key result
Parameter:
SI
Test group / Remarks:
0.5%
Remarks on result:
not measured/tested
Key result
Parameter:
other: cell count index
Value:
1.1
Test group / Remarks:
0.5%
Key result
Parameter:
other: ear weight index
Value:
0.99
Test group / Remarks:
5%
Key result
Parameter:
other: local lymph node weight index
Value:
1.26
Test group / Remarks:
5%
Key result
Parameter:
other: cell count index
Value:
1.38
Test group / Remarks:
5%
Key result
Parameter:
SI
Test group / Remarks:
5%
Remarks on result:
not measured/tested
Key result
Parameter:
other: ear weight index
Value:
0.97
Test group / Remarks:
50%
Key result
Parameter:
other: local lymph node weight index
Value:
1.3
Test group / Remarks:
50%
Key result
Parameter:
other: cell count index
Value:
1.2
Test group / Remarks:
50%
Key result
Parameter:
SI
Test group / Remarks:
50%
Remarks on result:
not measured/tested
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
The test substance LEE011-A1 exceeded the LN count thresholds at 5% without statistical significance when compared to vehicle. The highest dose did not follow the expected response to the dose therefore the LN weight data were used to calculate the threshold concentration.

EAR WEIGHT: LEE011-A1 did not cause any relevant changes in ear weight up to a concentration of 50% in dimethyl formamide.

LOCAL LYMPH NODE WEIGHT: LEE011-A1 exceeded the LN weight thresholds at 5% and 50% without statistical significance when compared to vehicle. LEE011-A1 did cause a clear dose reponse in LN weights.

CLINICAL OBSERVATIONS:
There were no clinical observations attributable to treatment with LEE011-A1.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

IRRITATION:
No irritation of the ears was noted after visual examination in the majority of animals. Brown test substance remnants on the dorsal surface of the ears of all animals treated at 50% (Days 1-3) did not hamper scoring for erythema. Visual examination of the nodes revealed that all the nodes of the experimental animals were considered normal in size when compared to the vehicle control group. No macroscopic abnormalities of the surrounding areas were noted in any of the animals.


Any other information on results incl. tables

Table 1. Results of LLNA

Group Item b.w. gain [g] (mean ± SD) Ear weight index Local lymph node (LN) weight index Cell count index
1 Dimethyl formamide -0.5 ± 0.5 1.00 [22.67] 1.00 [4.82] 1.00 [4.28]
2 0.5% DNCB 0.3 ± 0.5 1.09* 1.94** 2.41**
3 0.5% LEE011-A1 -0.2 ± 0.4 1.00 1.14 1.1
4 5% LEE011-A1 0.0 ± 0.9 0.99 1.26 1.38
5 50% LEE011-A1 0.3 ± 0.5 0.97 1.3 1.2
  Threshold conc. (%) n.a. n.a. 31.3 n.a.

Body weight gain (mean ± SD) was calculated from values of Day 4 before necropsy and Day 1 before treatment start. Ear weight in pairs data (apical area: 0.5cm²), weight and cell count data of LN pairs are represented as mean values (N=6) in relation to the control group 1 (index = 1). Values in brackets show mean values (weight in [mg], cell count in [E+06/mL]). n.a.: not applicable. *: P<0.05 treated groups vs control group 1, **: P<0.01 treated groups vs control group 1.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1B (H317) according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the modified LLNA, the test substance exceeded the LN weight threshold at 5% and 50% and the LN cell counts threshold at 5% without statistical significance. It did not cause relevant changes in ear weight up to a concentration of 50%. Therefore, the test substance is considered to be a weak sensitiser without irritating potential and has to be classified as Skin Sens. 1B (H317).

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